GSM4564438: BIN67 BRG1 ChIPseq IP Rep4; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
SMARCA4
Cell type
Cell type Class
Gonad
Cell type
BIN-67
NA
NA
Attributes by original data submitter
Sample
source_name
Cell Line
cell line
BIN67
transfected vector
BRG1
antibody
BRG1 abcam #ab110641
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP-seq was performed as previously described (Raab JR et. al. (2017) Epigenetics Chromatin). Briefly, cells (5e6 cells) were fixed for 30 minutes at 4°C in 0.3% methanol free formaldehyde, quenched for 5 minutes with 125mM glycine, washed 3 times and snap frozen in liquid nitrogen and stored at -80°C. Frozen pellets were thawed for 30 minutes on ice, resuspended in 1mL swelling buffer (25mM HEPES + 1.5mM MgCl2 + 10mM KCl + 0.1% Igepal containing 1mM PMSF and 1X protease inhibitor cocktail, Roche) and incubated 10 minutes at 4°C. Cells were dounced 20 strokes with a “B” pestle and then nuclei were pelleted at 2000 rpm for 7 minutes at 4°C. The nuclei were washed with 10mL MNase Digestion Buffer (15mM Hepes pH 7.9, 60mM KCl, 15mM NaCl, 0.32M Sucrose) and pelleted at 2000 RPM for 7 minutes at 4°C. The pellet was then resuspended in 1mL MNase Digestion Buffer per 4e7 cells + 3.3µL 1M CaCl2 per mL + PMSF (1mM) and Protease Inhibitor Cocktail (1X, Roche), then incubated for 5 minutes at 37°C to warm. We added MNase (NEB M0247S 2000U/µl) at 0.5µL/1e7 cells and incubated for 15 minutes at 37°C with agitation. Following digestion, the MNase was chelated using 1/50 volume 0.5M EGTA on ice for 5 minutes. We added 1 volume of 2X IP Buffer (20mM TrisCl pH 8, 200mM NaCl, 1mM EDTA, 0.5mM EGTA), then passed the sample through a 21G needle 5 times and added Triton X-100 to 1% final concentration. The sample was cleared at 13,000 RPM for 15 minutes at 4°C and chromatin was incubated with BRG1 antibody (abcam #ab110641) overnight at 4°C. Antibody/chromatin complexes were captured with protein A magnetic beads (Bio-Rad) for 2 hours at 4°C, washed 5 times with wash buffer (50mM Hepes pH 7.9/500mM LiCl/1mM EDTA/1% IGEPAL-ca-630/0.7% Na-Deoxycholate) and once with 10mM Tris/1mM EDTA/50mM NaCl. DNA was eluted at 65°C with agitation using 100µL freshly-prepared 1% SDS + 100mM NaHCO3. Crosslinks were reversed overnight by adding 5µL of 5M NaCl and incubating at 65°C. DNA was treated with 3µL RNaseA (30mg/mL) for 30 minutes at 37°C and then Proteinase K (20mg/mL) for 1 hours at 56°C and purified wit Zymo Clean and Concentrator ChIP Kit and quantified using Qubit before library preparation (Kapa Hyperprep). Libraries were sequenced on an Illumina NextSeq (Single End x 75 bp – 1 sample) and on an Illumina HiSeq4000 lane (Single End x 50 bp – 3 samples). Libraries were prepared with the Kapa Hyperprep kit following standard protocols.